Bone repair material preserving fluid for promoting bone cell differentiation and inhibiting bone hydatid growth

Abstract

The invention belongs to the technical field of biological material preservation, and particularly relates to a bone repair material preservation solution for promoting bone cell differentiation and inhibiting bone hydatid growth, the preservation solution takes PBS-CMCS gel and a high-glucose DEME culture medium as main effective components, the preserving fluid has the effects of maintaining high activity of osteoblasts, reducing apoptosis of bone cells, maintaining good biological activity of bone tissues and reducing degradation of bone matrixes, and does not destroy and dissolve inorganic substances (calcium) of the bone tissues, so that the mechanical properties of bone tissue repair materials can be maintained for a long time; the preservative solution is a preservative solution with biocompatibility and biological activity.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterial preservation, and specifically to a bone repair material preservation solution for promoting bone cell differentiation and inhibiting the growth of bone hydatids, and a preparation method thereof. Background Art

[0002] In clinical medical work such as orthopedics and traumatology, in order to maintain the speed of bone window fusion after surgery and reduce rejection reactions, the use of autologous bone materials to replant and repair tissue defects after surgery has become an increasingly common method. Therefore, the intact and activated preservation of bone tissue materials has become an important factor in the success of replantation and the degree of recovery of the patient.

[0003] At present, the methods for preserving bone materials are: the first is wet preservation using honey, alcohol, chlorhexidine, formalin, and glycerol as the preservation matrix. However, alcohol, formalin, and chlorhexidine preservation solutions will produce decalcification and damage bone tissue density. Honey, glycerol, and other biological preservation solutions have no sterilization and virus removal effects. The second is low-temperature preservation, which preserves bone materials in a deep low-temperature environment (-80°C). Before replantation, the freeze-dried bone is taken out 1 hour before surgery and rehydrated in Ringer's solution. Improper operation often causes fracture of bone transplant materials, and accidental damage caused by excluding operational factors is also difficult to avoid; at the same time, cumbersome operations can easily cause bone material contamination, leading to bone replantation failure. The third is freeze-drying and room temperature storage. It is only applicable to some bone materials that are allowed to be freeze-dried and preserved after decalcification or deproteinization, and there is also the defect of decreased biological activity and mechanical strength of bone materials.

[0004] Therefore, it is necessary to find a long-term preservation method that does not involve deep hypothermia. Summary of the invention

[0005] In view of the above technical problems, the present invention intends to use DMEM culture medium as the basic preservation medium, add PGS-CMCS gel, and reasonably match them according to certain concentrations and proportions, thereby significantly improving the high activity of osteoblasts during the preservation process and achieving the effect of rapid attachment of osteoblasts to rewarmed bone materials.

[0006] The inventor team has conducted a series of studies on the promotion of osteogenic differentiation by polysebacic acid glycerol. In the related studies on the preparation of micro-patterned structures to regulate the differentiation of stem cells, it was found that over-swollen (swelling rate ≥ 100%) polysebacic acid glycerol showed a trend of transformation to sol in physiological saline. Since the surface etching micro-pattern disappeared, it had basically no positive effect on osteogenic differentiation. However, in the presence of solid hard materials such as bone, hydroxyapatite / hydroxyapatite-like, polysebacic acid glycerol significantly promoted the adhesion and proliferation of osteoblasts on the bone surface and the differentiation of stem cells. Furthermore, it was found that under long-term (30 to 120 days) low-temperature storage, osteoblasts co-cultured with bone materials still maintained high activity. Following this idea, the present invention prepared a bone repair material preservation solution containing PGS-CMCS gel to promote osteocyte differentiation.

[0007] Specifically, the technical solution of the present invention is as follows: A bone repair material preservation solution for promoting bone cell differentiation and inhibiting the growth of bone hydatid cysts comprises 20-30 g / L PGS-CMCS gel, high-sugar DEME culture medium (D-glucose concentration is 4500 mg / L) and dihydroartemisinin; the PGS-CMCS gel is obtained by mixing polysebacic acid glyceride with carboxymethyl chitosan and then soaking in physiological saline; the mass ratio of the polysebacic acid glyceride to carboxymethyl chitosan is 3-5:1.

[0008] Furthermore, the concentration of the PGS-CMCS gel is 20-25 g / L; the pH of the bone repair material preservation solution that promotes osteocyte differentiation is 7.10-7.40.

[0009] Furthermore, the bone repair material includes autologous or allogeneic transplanted bone, bionic bone, or cartilage transplant with a fixed shape.

[0010] The present invention also provides a method for preparing a bone repair material preservation solution for promoting bone cell differentiation and inhibiting the growth of bone hydatids, comprising the following steps: S1: Sebacic acid and glycerol are melt polymerized at a molar ratio of 1:1.5-2 to obtain polyglycerol sebacate, which is then mixed with carboxymethyl chitosan at a mass ratio of 3-5:1, and the mixture is soaked in an equal mass of physiological saline to obtain PGS-CMCS gel; S2: Use a syringe to extract PGS-CMCS gel, inject it into the stirring high-sugar DEME medium, add dihydroartemisinin to make the final concentration of 40μmol / L~80μmol / L, continue stirring for 5~10 minutes, adjust the pH to 7.10~7.50, and store in bags.

[0011] Furthermore, the same effect as the PGS prepared by the present invention can be achieved by soaking polyglycerol sebacate with an average molecular weight within 5000-20000 in a mixed solution of glycerol and physiological saline.

[0012] Furthermore, the bone repair material preservation solution of the present invention, which promotes osteocyte differentiation and inhibits the growth of hydatid cysts, has the effect of promoting the differentiation of stem cells into osteocytes and can be used as an osteogenic differentiation culture medium.

[0013] The present invention also provides an application of the above-mentioned bone repair material preservation solution for promoting bone cell differentiation and inhibiting bone hydatid growth in preserving bone repair materials, the application comprising: placing the bone repair material preservation solution for promoting bone cell differentiation into a container and stirring it at a speed of 10-15 r / min for 2-3 minutes, washing the bone repair material with sterile saline, soaking it in the bone repair material preservation solution for promoting bone cell differentiation, and storing it at 0-4°C after sealing.

[0014] The beneficial effects of the present invention are as follows: the present invention provides a bone repair material preservation solution that promotes bone cell differentiation and inhibits the growth of bone hydatids. The preservation solution has the functions of maintaining high activity of osteoblasts, reducing osteocyte apoptosis, maintaining good biological activity of bone tissue, and reducing bone matrix degradation. At the same time, the preservation solution has no destructive and dissolving effect on inorganic matter (calcium) in bone tissue, so the mechanical properties of bone tissue repair materials can be maintained for a long time. The present invention is a preservation solution with both biocompatibility and biological activity.

[0015] In addition, the mixed gel (PGS-CMCS gel) formed by interweaving carboxymethyl chitosan with mixed polysebacic acid glyceride gel formed by polymerization of excess glycerol significantly improves the various properties of the preservation solution, especially the antibacterial property. Carboxymethyl chitosan promotes the water solubility of PGS and forms a cross-linked network with PGS, acting as a carrier and solubilizing agent. At the same time, the mixture has a sustained-release effect on carboxymethyl chitosan to maintain the antibacterial effect for a longer time. Compared with the bone repair material preservation solution with DMEM culture medium as the main component, the preservation solution of the present invention has lower cost and is more convenient to prepare. The osteoblast co-culture experiment confirmed that when bone marrow mesenchymal stem cells are co-cultured with bone repair materials, the preservation solution helps to promote bone cell differentiation.

[0016] By adding dihydroartemisinin to the bone repair material preservation solution, the soaked and preserved bone repair material can be specifically used to repair bone defects caused by hydatid cysts. DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand the present application, the present application is further explained in conjunction with specific implementation methods.

[0018] In recent years, polyglycerol sebacate (PGS) has received widespread attention due to its many excellent properties. A large number of basic scientific studies and animal experiments have also confirmed that it is suitable for tissue engineering, and related reports have confirmed that it can effectively promote bone regeneration in vivo. On this basis, the present invention considers promoting the solubility of PGS and selects chitosan as an antibacterial agent and cosolvent. Chitosan has good cell affinity but poor water solubility, but a chitosan derivative, carboxymethyl chitosan, has good water solubility and can destroy the cell membrane structure of microorganisms, causing leakage of cell contents, thereby achieving a bactericidal effect. In the medical field, carboxymethyl chitosan has been often used to prepare antibacterial dressings to accelerate wound healing.

[0019] In some embodiments of the present invention, sebacic acid and glycerol purified by recrystallization are mixed in a molar ratio of 1:1.5-2, melted at 130-140°C, stirred for 16-32h with N2, then vacuumed at 130-140°C for 24-48h, and cooled to room temperature to obtain PGS. PGS with different polymerization degrees show a sol-gel-high elastomer transition. In the case of excess glycerol polymerization, the PGS polymer of the present invention shows a paste between sol and gel.

[0020] In some embodiments of the present invention, PGS and carboxymethyl chitosan are administered separately to achieve a bone material preservation effect, specifically including the use of a bone material preservation solution with PGS and high-glucose DMEM culture medium as main components, a bone material preservation solution with carboxymethyl chitosan and high-glucose DMEM culture medium as main components, and a bone material preservation solution prepared by sequentially adding PGS and carboxymethyl chitosan to a high-glucose DMEM culture medium, or a bone material preservation solution prepared by sequentially adding carboxymethyl chitosan and PGS to a high-glucose DMEM culture medium, all of which have a certain bone material preservation effect.

[0021] In the embodiment of the present invention, GraphPad prism 8.0 statistical software was used for statistical analysis, and the data were expressed in the form of mean ± standard deviation (mean ± SD). Independent sample t test (student's test) and one-way ANOVA (one-way ANOVA) were used to compare two or more groups of data. Two-way ANOVA (two-way ANOVA) was used to compare data from different groups and different time points. p < 0.05 means that the difference is statistically significant. The p value is embodied in the picture as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: p > 0.05.

[0022] Example 1 Preparation of PGS-CMCS gel: The recrystallized and purified sebacic acid and glycerol were mixed in a molar ratio of 1:2, melted at 130°C, stirred with N2 for 24 hours, and then vacuumed at 130°C for 48 hours, cooled to room temperature to obtain polysebacic acid glycerol ester. 20g of polysebacic acid glycerol ester and 5g of carboxymethyl chitosan were soaked in an equal mass of physiological saline for 2 hours to obtain PGS-CMCS gel.

[0023] A bone repair material preservation solution for promoting bone cell differentiation and inhibiting the growth of bone hydatids Stir the PGS-CMCS gel for 5 min, extract 20 g of PGS-CMCS gel with a syringe, inject it into 1000 ml of stirring high-sugar DEME medium (Prunosai PM150210), continue stirring for 10 min, add dihydroartemisinin to a final concentration of 80 μmol / L, adjust the pH to 7.10-7.50, and store in bags.

[0024] This embodiment provides a method for using a bone repair material, comprising the following steps: Bone tissue materials were prepared, pretreated, and divided into ten groups. The bone repair material preservation solution that promotes osteocyte differentiation was placed in a container and stirred at a speed of 10-15 r / min for 2-3 minutes to keep the PGS-CMCS gel in a uniform suspension state. The bone repair material was washed with sterile saline and immersed in the bone repair material preservation solution that promotes osteocyte differentiation. After standing, the PGS-CMCS gel partially precipitated and wrapped the bone repair material, which was then sealed and stored at 4°C.

[0025] The preservation solution is used to preserve bone tissue materials from the time they are taken out of the body to the time they are replanted in the patient's body, ensuring that they are sterile and virus-free during the preservation period. Furthermore, ten groups of samples were preserved for 30 to 120 days (4°C), and samples of the preservation solution were taken at 30, 60, 90, and 120 days for bacterial culture and virus stock testing. This confirms whether the preservation solution is infected with microorganisms such as bacteria and fungi and the presence of virus cells.

[0026] When sampling, use a syringe to extract the preservation liquid in the preservation bag, and use an external patch to hot-melt bond or physically bond to keep it sealed.

[0027] Bacterial culture and virus test results: Samples of the preservation solution were taken at 30, 60, 90, and 120 days for three-day culture observation of Candida albicans, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Chloramine Bacillus, and no bacteria were detected. The presence of tumor cells, influenza virus, etc. was tested, and no viral cells were found, which shows that the bone repair material preservation solution of the present invention has the effect of killing viruses and bacteria.

[0028] Example 2 Comparative test: At room temperature, the bone repair material preservation solution was prepared in the following manner and divided into four groups: Group A: bone repair material preservation solution prepared in the same manner as in Example 1; Group B: 25 g of the polysebacic acid glyceride prepared in Example 1 was soaked in an equal mass of physiological saline for 2 h to obtain PGS gel, and a bone repair material preservation solution without CMCS was prepared, and the bone repair material was preserved in the same manner as in Example 1; Group C: 25 g of carboxymethyl chitosan was soaked in an equal mass of physiological saline for 2 h to obtain CMCS gel, and a bone repair material preservation solution without PGS was prepared. The bone repair material was preserved in the same manner as in Example 1; Group D: 20 g PGS and 5 g CMCS were soaked in equal amounts of physiological saline for 2 h, 17.5 g PGS gel and 2.5 g CMCS gel were extracted with a syringe, and injected into 1000 ml high-sugar DEME medium under stirring, and stirred for 10 min. The bone repair materials were preserved according to the method of Example 1; Control group: Bone material was freeze-dried at -80°C, taken out during sampling, rehydrated in Ringer's solution, and added with high-glucose DEME medium as the control group.

[0029] Table 1 Univariate variables of each group Component / Group Group A Group B Group C Group D PGS-CMCS gel Mixed gel state / / Join PGS and CMCS in turn PGS Gel / Equal quality PGS gel / / CMCS gel / / Equal quality CMCS gel / Solution pH 7.2 7.2 7.3 7.2 Obtaining bone material: Fifteen healthy New Zealand white rabbits weighing 2kg to 2.2kg were selected. The upper limbs were disinfected, and a longitudinal incision of 25mm was made along the anterolateral side of the upper limbs. The radius was cut, separated and exposed layer by layer. A 10mm osteotomy was performed at the middle and upper third of the radius. The attached soft tissue, cartilage and bone edge burrs were removed, and the rabbits were rinsed with saline to obtain bone material.

[0030] Take the bone material storage bag, put the bone samples in according to the identification number, and divide the 12 bone materials into 5 groups, three in each group, and store them in a refrigerator at 0~2℃ according to the treatment methods of group A, group B, group C, and group D. The control group is deep frozen at -80℃. (Among them, samples that failed to work or precipitated due to storage problems below -5℃ were not recorded in this study).

[0031] After 120 days of storage, the cells were taken out and thawed, stored at 4°C for three days, and then co-cultured with osteoblasts for 3-7 days for cell activity detection.

[0032] Example 3 1. Component analysis: Four groups of samples were taken at 30, 60, 90 and 120 days, and multi-site element content analysis was performed on a scanning electron microscope. The inorganic substance content was shown in the following table: Table 2 Element contents of bone materials in each group Element content / time (day) O（%） Ca (%) P（%） S（%） 30 35.56±1.32* 24.22±1.38* 10.47±1.76* 0.10 60 34.32±1.46* 21.82±1.73* 9.77±1.19* 0.05 90 32.34±1.59* 19.69±1.81* 9.47±2.03 0.12 120 30.30±2.87* 19.70±1.20* 8.26±1.04* 0 The above data show that under long-term storage (two months), the contents of elements such as Ca, P, and O in the samples are similar to the contents of bone tissue components in the body, indicating that the preservation effect is good and no decalcification or loss of other elements occurs.

[0033] 2. Bone material morphology detection: While performing multi-site element content analysis on a scanning electron microscope, the gross morphology of the bone material and the porosity of the bone cavity were marked and counted. The results are as follows: Table 3 Comparison of pore size and porosity results Group Pore ​​size (μm) Porosity (%) Group A 365.81±7.34 50.10±0.73 Control group 361.93±8.58 50.53±0.92 Morphological observation showed that the bone was in normal condition, maintaining the original interstitial structure, with uniform interstitial spaces in the inner lumen. Visual observation showed that the preservation solution was translucent in color, with no turbid precipitation except for the mixed gel. The above results showed that the preservation solution of the present invention had no destructive effect on the pore structure of the bone material.

[0034] 3. Mechanical properties testing: Three pieces of bone materials from group A and control group were taken for torsional strength test on a universal tensile machine. The results showed that the maximum torsional strength of the bone materials from group A was 8.17±0.91 N·m, and its torsional resistance was much higher than that of the freeze-dried bones in the control group (4.69±1.03 N·m).

[0035] 4. Cell co-culture experiment: Bone marrow mesenchymal stem cells and osteoblasts were cultured in a culture plate with the bone materials (preserved for 120 days) stored in Group A, Group B, Group C, Group D and the control group in Example 2 of the present invention for 7 days. The freeze-dried bone materials were used as a control. The cell material complexes were taken from each group and the alkaline phosphatase activity of osteoblasts was detected. The results are shown in Table 2.

[0036] Table 4 ALP changes after co-culture of osteoblasts with different groups of preservation solutions of the present invention Group Alkaline phosphatase activity (U / L) Group A 0.983±0.045* Group B 0.832±0.037* Group C 0.871±0.082* Group D 0.893±0.078* Control group 0.756±0.042 The ALP content shows that the PGS-CMCS gel of the present invention has a better effect on promoting osteoblast proliferation than the use of PGS or CMCS alone. Compared with the freeze-dried bone of the control group, the preservation solution of the present invention can better maintain the biological activity of the bone material and help cell adhesion and proliferation.

[0037] 5. Effect of inhibiting hydatid cyst Bone repair material preservation solution containing 10μmol / L, 20μmol / L, 40μmol / L, and 80μmol / L of dihydroartemisinin was cultured with protoscolex in vitro, and the survival status and morphological changes of multilocular Echinococcus protoscolex were observed under a microscope on the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th days of culture. The results showed that there were no surviving multilocular Echinococcus protoscolex at 40μmol / L, and the bone repair material preservation solution had an inhibitory and killing effect on the in vitro growth of multilocular Echinococcus protoscolex.

Claims

1. A bone repair material preservation solution for promoting bone cell differentiation and inhibiting the growth of bone hydatids, characterized in that: The method comprises 20-30 g / L PGS-CMCS gel and high-sugar DEME culture medium; the PGS-CMCS gel is obtained by mixing polysebacic acid glyceride with carboxymethyl chitosan and then soaking in physiological saline; the mass ratio of the polysebacic acid glyceride to carboxymethyl chitosan is 3-5:1; Also included is dihydroartemisinin.

2. The bone repair material preservation solution for promoting bone cell differentiation and inhibiting the growth of bone hydatids as claimed in claim 1, characterized in that: The concentration of the PGS-CMCS gel is 20-25 g / L; the pH of the bone repair material preservation solution that promotes osteocyte differentiation is 7.10-7.

40.

3. The bone repair material preservation solution for promoting bone cell differentiation and inhibiting the growth of bone hydatids as claimed in claim 1, characterized in that: The bone repair material includes autologous or allogeneic transplanted bone, bionic bone, and cartilage transplant with a fixed shape.

4. A method for preparing a bone repair material preservation solution for promoting bone cell differentiation and inhibiting the growth of bone hydatids, characterized in that: The steps include: S1: Sebacic acid and glycerol are melt polymerized at a molar ratio of 1:1.5-2 to obtain polyglycerol sebacate, which is then mixed with carboxymethyl chitosan at a mass ratio of 10-15:1, and the mixture is soaked in an equal mass of physiological saline to obtain PGS-CMCS gel; S2: Use a syringe to extract PGS-CMCS gel, inject it into the stirring high-sugar DEME medium, add dihydroartemisinin, continue stirring for 5-10 minutes, adjust the pH to 7.10-7.50, and store in bags.

5. The use of the bone repair material preservation solution for promoting osteocyte differentiation and inhibiting the growth of hydatid cysts as claimed in claim 1 in preserving bone repair materials, comprising: The bone repair material preservation solution that promotes osteocyte differentiation is placed in a container and stirred at a speed of 10-15 r / min for 2-3 minutes. The bone repair material is washed with sterile saline, immersed in the bone repair material preservation solution that promotes osteocyte differentiation, and then sealed and stored at 0-4°C.

6. Use of the bone repair material preservation solution for promoting bone cell differentiation and inhibiting the growth of hydatid cysts as claimed in claim 1 in preserving bone repair materials for repairing bone defects caused by hydatid cysts.